The aims of this research are to purify and characterize an erythroid enhancing activity (EEA) elaborated by an established human cell line, GCT. Large quantities of serum-free conditioned medium will be concentrated by ultrafiltration and fractionated by gel permeation, ion exchange chromatography, as well as newer techniques such as hydrophobic chromatography, affinity chromatography, high performance liquid chromatography (HPLC), reverse phase liquid chromatography (RPLC), isoelectric focusing and preparative polyacrylamide gel electrophoresis. In addition, we will study the effect of purified EEA on its target cell and clarify the role of accessory cells such as T cells or monocytes in erythropoiesis. This will be done by enriching for erythroid progenitor cells by isopycnic centrifugation, centrifugal elutriation and activiated cell sorting followed by selective depletion of T cells and monocytes by appropriate monoclonal antibodies. The cell line specificity of EEA will be tested on established human cell lines with erythroid (K562), myeloid (HL60) and lymphoid (RPMI 1788) properties. The role of EEA in early hematopoiesis will be investigated by studying its effects on progenitor cell survival in long term human bone marrow cultures. Factors that affect the mediator, will be studied in normal monocytes and transformed GCT cells. The long term objectives are to further our understanding of the rgulation of early erythropoiesis and specifically the role of EEA in the expansion of the erythroid progenitor cell compartment and the augmentation of fetal hemoglobin production, in sickle cell anemia, thalassemia and myeloproliferative diseases.